We previously found that insulin rapidly suppresses the activity of a soluble PTPase in skeletal muscle of insulin-sensitive subjects, but not in subjects resistant to insulin and that activity of a particulate PTPase is chronically elevated in muscle of resistant subjects. Because either of these abnormalities could be responsible for insulin resistance, we have begun to identify and characterize the PTPases in human skeletal muscle. Putative PTPase cDNA sequences were amplified by PCR from total RNA isolated from human muscle using Taq polymerase and degenerate oligonucleotide primers based on conserved amino acid sequences in known PTPases. PCR products of the expected size (340 bp) were purified by agarose gel electrophoresis and reamplified. The second round PCR product was purified and cloned. Analysis of 50 clones by either direct sequencing or by restriction enzyme analysis revealed 12 different PTPase-like sequences. Two of the sequences are unique and are represented by multiple clones. Two others are identical to previously described PTPases (PTP1B and TCPTP) and one is similar to a third PTPase (PTPH22) except for a single amino acid substitution. The amplified sequences occur at frequencies ranging from 2% to 38%. Five other clones differ from TCPTP by only one base. The 340 bp PCR product was used to probe a Northern blot of total RNA from human skeletal muscle. This revealed two mRNA bands of about 4.7 kb and 2.4 kb. Initial screening of a human skeletal muscle cDNA library with the 2 novel PCR products has identified several positive clones for each.